【作者】 赵莹；

【导师】 朱春玉；

【作者基本信息】 辽宁大学 ， 微生物学， 2021， 硕士

【摘要】 蓝莓溃疡病是由葡萄座腔菌(Botryosphaeria dothidea)导致的真菌性病害,可导致蓝莓大幅减产。本研究以葡萄座腔菌为靶标菌,以本实验室自主筛选获得的拮抗菌株链霉菌CX3(Streptomyces sp.)为研究载体,对其田间防效进行了评价,测定了该菌株全基因组,并对其功能进行了解析,对菌株发酵液的有效成分初步分离,通过研究苯丙烷代谢和水杨酸等途径物质含量和酶活性的改变探讨了该发酵液是否可以调控寄主植物来抵御蓝莓溃疡病菌的侵入。主要研究结果如下:1、链霉菌CX3全长8516989 bp,共编码基因7504个,GC含量72.73%。COG和KEGG等数据库注释结果表明链霉菌CX3中抗生素的生物合成及次生代谢产物的生物合成有关基因被大量富集。通过anti SMASH对CX3基因组的次级代谢产物基因簇进行预测,预测结果表明CX3有32个基因簇,能够参与合成细菌素、萜烯类、丁内酯、羊毛硫肽类和铁载体等多种化合物。其中化合物Dutomycin、Ficellomycin、Kirromycin、Lactonamycin和Nocathiacin均有抗细菌作用,ECO-02301具有较强的抗真菌活性。2、鉴于链霉菌CX3次级代谢产物基因簇的多样性,为进一步明确其产生的活性物质种类,对CX3发酵液的活性物质进行理化特性探究及初步分离鉴定。CX3发酵液活性物质的稳定性分析表明,其紫外稳定性强,热稳定性较好,耐碱不耐酸。纸层析实验结果表明,CX3发酵液中至少含有两种活性成分,其中一种活性成分可能是水溶性I型抗生素。Betina溶剂系统实验结果表明,活性成分不溶于乙酸乙酯和苯这两种有机溶剂,极性中等。纸电泳实验结果表明,这两种活性成分一种为中性,另一种为弱酸性。3、采用人工接种法,开展链霉菌CX3发酵液的田间防效实验,结果表明该发酵液具有良好的保护作用,达53.7%,明显优于供试的2种常用的化学农药。鉴于此,我们对CX3发酵液是否能够诱导寄主抗病性进行了测定,结果表明CX3发酵液显著提高了蓝莓叶片苯丙氨酸解氨酶和几丁质酶活性,并显著提升了宿主总酚、类黄酮、木质素、原花青素、水杨酸等抗菌物质的含量,激活了宿主体内苯丙烷代谢途径和水杨酸途径,从而提高了宿主的抗病性。综上,链霉菌CX3发酵液活性物质性质较为稳定,对蓝莓溃疡病具有较好的田间防治效果,同时能够激发苯丙烷代谢途径和水杨酸通路诱导蓝莓植株产生抗性抵御病原菌侵入,具有较好的开发价值和应用前景。更多还原

【Abstract】 Blueberry canker is a fungal disease caused by Botryosphaeria dothidea,resulting in a large yield reduction of blueberry fruit.In this study,B.dothidea was used as the target bacteria,and the antagonistic strain Streptomyces sp.CX3,which was independently screened by our laboratory,was used as the research carrier.Field experiments were carried out to evaluate its control effect,and the whole genome sequencing of Streptomyces sp.CX3 was performed to analyze the function.Then,preliminary isolation of antimicrobial active substances from Streptomyces sp.CX3 was also conducted,and the changes in the substance content and enzyme activity of phenylpropane metabolism and salicylic acid were studied to explore whether the fermentation broth can regulate the host plant to resist the invasion of blueberry canker bacteria.The research results are as follows:1.Whole genome sequencing of Streptomyces sp.CX3 based on the Illum ina platform showed that the genome was 8516989 bp after assembly,with 7504 coding genes,and GC content of 72.73%.The annotation results in COG and KEGG database showed that genes related to biosynthesis of antibiotics and biosynthesis of secondary metabolites were-largely enriched.The anti SMASH was used to predict the gene clusters of secondary metabolites in Streptomyces sp.CX3 genome,and the results indicated that 32 gene clusters was found,which were involved in the synthesis of bacteriocins,terpenes,butyrolactones,lanthiopeptides and siderophores.Among them,the compounds Dutomycin,Ficellomycin,Kirromycin,Lactonamycin and Nocathiacin all have antibacterial effects,and ECO-02301 has strong antifungal activity.2.Due to the diversity of the secondary metabolite gene clusters in Streptomyces sp.CX3,the physio chemical properties and preliminary separation and identification of the active substances in the Streptomyces sp.CX3 fermentation broth were investigated.The results showed that Streptomyces sp.CX3 fermentation broth had strong UV stability,good thermal stability,alkali resistance but not acid resistance.The results of paper chromatography showed that at least two active components were present in Streptomyces sp.CX3 fermentation broth,one of which might be water-soluble type I antibiotic.The results of Betina solvent system experiment showed that the active ingredient was insoluble in ethyl acetate and benzene,and had moderate polarity.The results of paper electrophoresis experiments showed that one of the active components was neutral and the other was weak acid.3.The artificial inoculation method was used to carry out the field control experiment of the fermentation broth of Streptomyces sp.CX3.The results showed that the fermentation broth has a good protective effect,reaching 53.7%,which is significantly better than the two commonly used chemical pesticides tested.In view of this,we determined whether Streptomyces sp.CX3 fermentation broth can induce host disease resistance.The results showed that Streptomyces sp.CX3 fermentation broth significantly increased the activities of PAL and CHI in blueberry leaves,and significantly increased the content of antibacterial substances such as total phenols,flavonoids,lignin,proanthocyanidins,salicylic acid,etc.,activate the phenylpropane metabolic pathway and the salicylic acid pathway in the host,thereby improving the host’s disease resistance.In conclusion,the fermentation broth of Streptomyces sp.CX3 are relatively stable,and has a good biocontrol effect on blueberry canker in field.Meanwhile,the fermentation broth can stimulate the phenylpropane metabolic pathway and the salicylic acid pathway to induce the disease resistance of blueberry plants indicating the promising development value and application prospects of Streptomyces sp.CX3.更多还原